In this study, scanning electron microscopy (SEM) and single-cell Raman spectroscopy (SCRS) were used to analyze the differences in the cellular morphology and intracellular macromolecular compounds of Lactobacillus delbrueckii subsp. bulgaricus ND02 in the normal and viable but non-cultivable (VBNC) states. The SEM results showed that the VBNC cells, with a wrinkled surface, were shorter and thicker than the normal ones. The Raman spectra of the VBNC cells were significantly different from those of the normal cells, mainly in terms of 12 peaks, such as 1673, 1650, 1459, 1030, and 1005 cm^-1, reflecting changes in intracellular biomacromolecules such as proteins and nucleic acids. It was shown that L. delbrueckii subsp. bulgaricus ND02 could adapt to adverse environments by regulating the metabolism of proteins, nucleic acids and lipids when induced to enter into VBNC state. The results also confirmed that single-cell Raman spectroscopy can allow non-destructive label-free detection of macromolecules inside normal and VBNC cells of L. delbrueckii subsp. bulgaricus ND02.

In order to obtain a comprehensive understanding of the trend of research on the viable bacteria but non-culturable (VBNC) state in bacteria, 836 pieces of research literature in this field published during the period from 1994 to 2021 retrieved from the Web of Science core collection database were comparatively analyzed in terms of the number of literature, publishing time, authors’ institutions, publication sources, research directions and keywords using bibliometrics. A knowledge map of keywords for visual analysis was obtained by Cite Space 5.8 software. The results showed that: from 1994 to 2021, the annual number of articles published on bacterial VBNC state showed a relatively stable growth trend, and the 836 articles, produced by 1528 research institutions in 67 countries or regions, are published in 355 publications; from 1994 to 2021, the top 10 countries produced 88.04% of the total literature volume; from 2011 to 2021, China ranked first in the number of publications in the world; and the number of research papers on bacterial VBNC state has always been at the forefront of the fields of microbiology, infectious diseases, biochemistry and molecular biology. The key words of Escherichia coli, VBNC, nonculturable state, survival and resuscitation are hot topics in the research area of bacterial VBNC state. In the future, more efforts are still needed in research on the VBNC state of bacteria, which will provide an important reference for food safety and the development and utilization of microorganisms.